There are numerous reasons to monitor hormone levels of an individual. Medically, physicians may choose, for example, to monitor progesterone and/or estrogen levels of women receiving hormone replacement therapy; androgen levels of men treated for prostate cancer; hormone levels of children with pituitary disorders; or progesterone levels of pregnant women. Sociologically, public officials and government agencies must address increasing concerns that chemicals in the environment, such as pesticides and fungicides, are affecting human health. Financially, professional sports organizations, charged with maintaining fair competition, routinely test athletes and racehorses for anabolic steroids which may influence the distribution of prizes.
Testing procedures currently used to detect anabolic steroids in professional athletes and racehorses require column chromatography to remove water, ions, and proteins from urine. The purified steroids are then chemically modified to make them more volatile for gas chromatography-mass spectrometry (GC-MS) analysis. An algorithm is used to search for 30-40 banned steroids based on CG retention time and two or three ions per molecule in the mass spectrum. This detection method is expensive and fallible. Automated purification systems, gas chromatographs and mass spectrometers are costly and technically complicated laboratory instruments that must be continually calibrated and operated by trained technicians in order to produce reliable results. Additionally, this method identifies only 30-40 of the literally hundreds of possible molecules in the steroid family. A person skilled in the chemical arts could easily derivatize a banned steroid to create a previously unknown or uncategorized molecule with potent physiological properties. The derivatized steroid would go undetected by the currently used method because the GC retention time and/or ions of the mass spectrum would not match those searched by the algorithm.
Quantitative determinations of anabolic steroids are complicated by the fact that steroids are ubiquitous in the human body. For example, urinanalysis for exogenous testosterone typically measures the ratio of testosterone to epitestosterone, but athletes wishing to beat a steroid test can simply counter their testosterone intake with a proportional intake of epitestosterone such that the ratio remains constant.
Steroids may also be detected through bioassay; however, currently available bioassays lack critical sensitivity and accuracy. For example, Raivio et al. (JCE&M, 86: 3, 2001, 1539) reported an assay for measuring androgen bioactivity in human serum. The assay consisted of a) a Gal4 DNA-binding domain operably linked to an androgen receptor ligand binding domain (Gal4-DBD:AR-LBD); b) a herpes simplex VP16 protein operably linked to the N-terminal region of the androgen receptor (VP16:AR(N terminus)), c) a luciferase reporter gene and d) an AR-interacting protein 3 (ARIP3) for amplification. Unfortunately, the constructs were incapable of detecting steroids with sufficient sensitivity or specificity to be useful as an assay.
Paris and colleagues (J. Clin. Endocrinol. Metab. 87: 2002, 791) also developed a bioassay with virtually no diagnostic utility. They reported an estrogen bioassay that employed recombinant HeLa cells expressing the estrogen receptor with an estrogen response element driving a luciferase reporter. However, HeLa cells contain endogenous aromatase, an enzyme that converts testosterone to estrogen. Although aromatase inhibitors may be added to the cells in an attempt to eliminate the endogenous conversion of testosterone, it is unconfirmed whether or not such an assay would provide a reliable means of detection.
Balasubramanian and Morse (Mol. Cell Biol. 19: 2977-2985) reported a tripartite construct (LexA-DBD:ER-LBD:VP16) in yeast useful in their studies of transcriptional activators. However, the use of a yeast based system and construct in a clinical diagnostics setting for the detection and monitoring of various steroids was not contemplated, presumably due to inherent challenges created by other components in serum, such as metals, sugars, and amino acids that affect yeast growth or the ability to detect the downstream reporter. Attempts to remove such factors, such as by extraction, can also bias the sample and make the results unreliable. Use of a yeast system in a bioassay also has inherent challenges due to endogenous properties of the yeast cells themselves, such as bioconversion of the steroids being assayed and factors affecting transcription efficiency, such as cross-reactivity issues, transport issues, and transcriptional issues.
A simple method of measuring steroids in a clinical setting is greatly needed. Currently, there are no affordable, sensitive, reliable and versatile methodologies capable of detecting steroids or other molecules capable of binding a steroid hormone receptor.